Cystic fibrosis is a lethal genetic disease afflicting approximately 30,000 individuals in the United States. Approximately 1 in 2500 Caucasians is born with the disease, making it the most common lethal, recessively inherited disease in that population.
Cystic fibrosis affects the secretory epithelia of a variety of tissues, altering the transport of water and salt into and out of the blood stream. In particular, the ability of epithelial cells in the airways, pancreas and other tissues to transport chloride ions, and accompanying sodium and water, is severely reduced in cystic fibrosis patients, resulting in respiratory, pancreatic and intestinal ailments. The clinical manifestation of cystic fibrosis is respiratory disease, characterized by airway obstruction due to the presence of a thick mucus that is difficult to clear from airway surfaces. This thickened airway liquid results in recurrent bacterial infections and progressively impaired respiration, eventually leading to death.
Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The coding regions of CFTR are composed of 27 exons dispersed over 250,000 base pairs (250Kb) of genomic DNA. During transcription, introns are spliced out and exons are joined together to form a 6100-bp mRNA transcript that is translated into the 1480 amino acid sequence of CFTR protein
The normal CFTR protein is a chloride channel protein found in membranes of cells that line the passageways of the lung, pancreas, colon and genitourinary tract. The CFTR protein is made up of five domains: two membrane-spanning domains (MSD1 and MSD2) that form the chloride ion channel, two nucleotide-binding domains (NBD1 and NBD2) that bind and hydrolyze adenosine triphosphate (ATP), and a regulatory (R) domain.
Protein synthesis normally involves interactions between amino acid side chains that results in folding of the protein into a thermodynamically preferred three dimensional structure. Proper protein localization within the cell is dependent on the protein forming the correct conformation. Some proteins appear to require interaction with other molecules in order to fold properly. Such molecules include proteins referred to as “molecular chaperones.” Chaperones stabilize newly synthesized polypeptides until they are assembled into their proper native structure.
The most common cause of cystic fibrosis which accounts for 70% of all cystic fibrosis cases results from a three nucleotide deletion that results in deletion of phenylalanine residue 508 (ΔF508 CFTR). The ΔF508 mutation occurs in the nucleotide sequence that codes for the first nucleotide-binding domain (NBD1) and results in retention of the mutant protein in the endoplasmic reticulum (ER) and subsequent degradation by the ubiquitin-proteosome pathway. The mechanism responsible for ER retention of the ΔF508 CFTR protein includes associations with cytoplasmic chaperones such as Hsc/Hsp70 and Hdj2.
One recently developed approach for the transfer of various cargo to cells involves the use of novel cell-targeting ligands, which increase the rate and specificity for the transport of molecules. Such ligands are also known as protein transduction domains (PTDs). The first protein discovered having such transduction properties was the HIV transactivator protein, TAT. See Green & Lowenstein, Cell, 55:1179-1188 (1988); Frankel & Pabo, Cell 55:1189-1193 (1988). Subsequently, an 11 amino acid transduction domain in TAT (TAT-PTD) responsible for the observed transduction properties was identified, based on its high basic residue content. See Fawell et al., Proc. Natl. Acad. Sci. USA 91:664-668 (1994). It has been shown that fusion protein constructs containing TAT-PTD are capable of delivering proteins to a wide spectrum of cell types both in vitro and in vivo. See Nagahara et al., Nat. Med. 4:1449-52 (1998); Vives et al., J. Biol. Chem. 272:16010-17 (1997); Shwarze et al., Science 285:1569-72 (1999); Vocero-Akbani et al., Nat. Med. 5:29-33 (1999); Moy et al., Mol. Biotechnol. 6:105-13 (1996). Other peptides having translocating properties include, for example, penetratins (Derossi et al., 1998, Trends in Cell Biology 8:84-87), Drosophila Antennapedia homeodomain, the cell attachment motif of foot and mouth disease virus (FMDV) (Villaverde et al., 1998, Biotechnology and Bioengineering 59:294-301); and VP-22 (Elliot & O'Hare, Cell 188:223-233 (1997)).
PTDs can be used to deliver full length proteins as well as small peptides. The advantage to protein transduction appears to be the efficiency of delivery and may be used to deliver cargo to treat various acute diseases, as well as chronic diseases, both genetic and acquired.
The efficiency of PTDs is apparently linked to its mechanism of action. PTDs interact electrostatically with anionic elements, such as GAGs, on the cell surface. These contacts draw the PTDs in close proximity to the plasma membrane where, by one or more unknown mechanisms, including, in all likelihood, endocytosis where the PTDs and their cargoes are delivered into the cell. Thus, because of the electrostatic interactions, the length and degree of charge within the PTDs can modify its efficiency. Therefore, longer peptides (10 and 12 mers) are better able to mediate transduction in GAG-deficient lines than short PTDs, although short PTDs (4 and 6-mers) still possess an intrinsic capacity for protein transduction, provided they can bind to the cell surface via interactions with charged dextran polymers.
Current treatments for cystic fibrosis generally focus on controlling infection through antibiotic therapy and promoting mucus clearance by use of postural drainage and chest percussion. However, even with such treatments, frequent hospitalization is often required as the disease progresses. New therapies designed to increase chloride ion conductance in airway epithelial cells have been proposed, but their long-term beneficial effects have not been established and such therapies are not presently available to patients.
Accordingly, improvements are needed in the treatment of cystic fibrosis. The present invention fulfills this need and further provides other related advantages.